Detection and identification of pathogenic microorganisms is an area of high concern in many areas of public health. Particularly important is the identification of pre-existing or newly arising strains of infectious agents such as bacterial, viral, or other disease causing organisms. Diagnostic efforts are hampered by either the total inability or length of time required to identify the presence or absence of a pathogenic organism in the host.
Since the early 2000s, methods of virus and other infectious agent detection have rapidly advanced. A primary detection mechanism for influenza virus, for example, is real-time PCR(RT-PCR) that provides sensitive and relatively rapid identification of a suspected strain of virus. Some of the advantages of RT-PCR are high sensitivity, high specificity, rapid time-to-result, scalability, cost, and quantitative nature. Sample type is less restricted with RT-PCR allowing for lower expense and more rapid viral identification than traditional methods. A severe shortcoming of PCR-based identification techniques is that they require known sequence information for effective detection. For example, RT-PCR uses probes that hybridize to a known sequence. If a significant mutation occurs in a viral target, the known effective probes are rendered useless as they will be unable to hybridize to a mutant sequence. The restricted sequence detection of standard PCR techniques also prevents rapid detection of naturally or intentionally inserted new virulence sequences that may either render the virus or bacteria resistant to therapy, or increase infectivity or transmissibility. Importantly, PCR techniques are unable to detect a previously unknown or unsuspected infectious agent in a sample.
The ability to detect and identify infectious agents is an essential first step in the assessment of biothreats. However, meaningful assessment of the risks posed by an infectious agent once detected, and the implementation of an effective means to mitigate the threat requires information like antibiotic resistance, toxin production, or virulence that current analytical systems are not designed to produce. Protein analyses can provide information useful for both the phylogenetic identification of microorganisms and the assessment of physiological cell functions, which are actuated by proteins. For instance, the amino acid sequence of structural proteins (e.g., ribosomal proteins) can provide information for phylogenetic identification, while amino acid sequences of functional proteins (e.g., toxins) can provide information on virulence. Unfortunately, prior art detection and identification methods that are able to use proteomic information require long analysis times and a prior understanding of the type of infectious agent that may be present in a sample.
Accordingly, there is a need to develop methods for rapidly detecting microorganisms, particularly bacterial organisms that permits protective measures or countermeasures to be quickly implemented in the event of an attack with weapons employing the same or as a robust method of identifying the causative agent(s) in an illness outbreak. Moreover, the demand for methods and assays capable of rapidly detecting and identifying microorganisms has applications beyond those of the military such as in the pharmaceutical, medical, food and public safety industries, and the like.